Recent studies suggest that signaling through cannabinoid receptor type 2 (CB2) on immune cells may play an important regulatory role in the generation and expression of host immunity. However, research into the distribution and function of CB2 has been limited by a lack of reagents to identify this receptor at the protein level. In particular, no monoclonal antibodies (mAbs) have been developed to detect CB2 protein in ive cells. Monoclonal Abs provide a reproducible and flexible reagent for tagging cell surface receptors, characterizing expression levels, and linking receptor expression to other cell features and functions. Monoclonal Abs can also provide a highly selective approach for neutralizing receptor function. The primary goal of this Phase I Cutting-Edge Basic Research Application (CEBRA) is to use novel cellular and molecular approaches to generate anti-CB2 mAbs directed against the extracellular sequences of CB2 and to validate their capacity to identify and neutralize CB2 expression by immune cells. The following two specific aims are proposed: 1) to employ a vector-based immunization protocol in CB2 receptor knockout mice to generate mAbs directed against cell surface epitopes of human CB2, and 2) to validate the capacity for selected mAbs to identify and/or neutralize CB2 expression on target immune cells. Several obstacles that may prevent the generation of desirable anti-CB2 mAbs will be addressed. CB2 receptor knockout mice, which lack expression of native CB2, will be used as an innovative strategy for bypassing self-tolerance and improving immune responsiveness. We have also prepared a lentiviral vector that expresses human CB2 cDNA that will be used in a prime-boost vaccination protocol to induce high anti-CB2 Ab liters. In addition, we will use Chinese hamster ovary (CHO) cells that have been transduced to express high levels of human CB2 as a target cell for identifying CB2 antibodies directed against the extracellular domains. With these aims and novel approaches, this Phase I CEBRA grant will develop important new mAb reagents that will facilitate future studies into the expression, regulation and function of CB2 receptors in immune cells as part of a Phase II CEBRA grant. These reagents will also facilitate research by other investigators interested in CB2, and provide a vaccination and mAb screening protocol that can be adapted to generate other mAbs, including mAbs directed against CB1. This work has the capacity to significantly accelerate cannabinoid research. [unreadable] [unreadable] [unreadable]